Researchers at the Federal University of Pelotas, Brazil, recently completed a study identifying circulating cell-free fetal DNA (ccffDNA) to determine fetal gender in pregnant mares. While ccffDNA has already been explored in humans, this is the first study to successfully demonstrate its presence ccffDNA–and thus aid in sex determination–in horses.Generally, veterinarians determine fetal sex-typing via transrectal or transabdominal ultrasound of the mare. In the current study, researchers isolated ccffDNA in the blood plasma of 20 Thoroughbred mares in their final three months of pregnancy.
The current study was conducted in two steps:

Step 1 involved a polymerase chain reaction (PCR) assay to detect ccffDNA in the plasma of pregnant mares. PCR determines sex at the molecular level by detecting the presence or absence of Y chromosome. The expressed sex-determining SRY gene suggests a male, while the absence of SRY indicates female gender.

The first stage PCR/SRY analysis produced an overall accuracy of 85%, with the second stage of analysis after reamplification achieving 95% accuracy. Researchers then confirmed the test results after foaling.
While this relatively noninvasive procedure proved successful in determining a foal’s gender in this study, the technique also opens the door for future prenatal detection of genetic diseases. The research team hopes to increase the test’s sensitivity for use in early pregnancy.

“We are working on a project to identify the minimum time to determine fetal DNA in the blood of mares,” explained researcher Tiago Collares, PhD, DVM. “In this sense, we could intervene in high-risk or genetically undesirable pregnancies.
“Our study was simple and innovative,” he continued. “Several studies in humans are new frontiers for studies in animals, especially horses.”
This study, “Equine fetal sex determination using circulating cell-free fetal DNA (ccffDNA),” was published in the February 2012 issue of Theriogenology. The abstract is available on Pubmed.